The present invention relates to a system for analyzing substances by using capillary zone electrophoresis (CZE) and, more particularly, to CZE systems using a multireflective cell.
When determining the composition of a substance through light absorption detection, photons are passed through the substance; that portion of energy not absorbed is thus detected. Energy spectra not absorbed by the substance can be used for at least two purposes: (1) to indicate the composition of an unknown substance; and (2) to determine the presence and/or quantity of a predetermined material (e.g., element, molecule, etc.) in the substance.
Capillary zone electrophoresis (CZE) is such a system for performing this analysis. To assess a substance, a sample is drawn through a long, transparent capillary by the process of electrophoresis (the movement of suspended particles through a fluid via an electromotive force). Electromagnetic energy (e.g., a laser beam or ultraviolet light) is passed through the transparent capillary. Energy is partially absorbed by the sample within the capillary. The energy not absorbed by the sample is detected by a photodetector. The detection can be performed whether the sample in the capillary is stationary or moving.
Detection is one of the most important areas of CZE. Various principles, such as those employed in spectrophotometric, mass spectrometric, electrochemical and radiometric detection, have been applied to CZE. Although the sensitivity of UV-visible absorption detection is perhaps the least among these detection methods, UV detectors are the most frequently used in CZE because of their simplicity and versatility.
Most CZE systems employ a single-pass detection technique, wherein electromagnetic energy passes once through the sample, and the light beam travels normal to the capillary axis, crossing the capillary only a single time. According to Beer's law, EQU A=.epsilon..multidot.b.multidot.c
where A is absorbance, .epsilon. is molar absorptivity of the sample, b is path length of the electromagnetic energy and c is sample concentration.
The CZE technique is successful only when the sample is thin enough for a sufficient dissipation of heat. Thus, CZE analysis is conducted on a small scale (e.g., a sample is typically 50-75 .mu.m thick). In single-pass detection, sensitivity is minimal because the path length is limited by the thickness of the sample. Therefore, even with state-of-the-art detectors, concentration detection limits are rarely lower than 10.sup.-6 M.
For a given molecule in a particular separation environment, absorption detection limits can be improved by decreasing the noise and/or increasing the path length. Some UV detectors for CZE have noise levels as low as 2.times.10.sup.-5 au, resulting in detection limits on the order of 10.sup.-6 M. The detection limit can also be improved by increasing the effective path length. In "Rectangular Capillaries for Capillary Zone Electrophoresis", Tsuda, T.; Sweedler, J. V.; Zare, R. N. Analytical Chemistry 1990, 62, 2149-2152, rectangular capillaries have been investigated for CZE. Using the rectangular capillary, a 20-fold increase in path length and a 15-fold increase in sensitivity was obtained. The increase in detection sensitivity was due to the wider dimension.
A "z"-shaped absorption cell has also been developed for capillary liquid chromatography, which significantly enhances path length and detection sensitivity. This seems promising, but the volume of the z-shaped cell is too large for CZE.
Axial-beam absorption detection was introduced in "Axial-Beam On-Column Absorption Detection for Open Tubular Capillary Liquid Chromatography", Xi, X.; Yeung, E. S. Analytical Chemistry 1990, 62, 1580-1585. A beam of light was introduced into a capillary along its major axis. The light beam struck the sample within the capillary. Light that was not absorbed exited from the end of the capillary and was detected. Axial-beam detection requires precise alignment of the capillary and the light source; it also restricts the choice of mobile phase to solutions with refractive indices higher than that of fused silica.
The "White cell" is referred to in "Spectroscopic Methods For Air Pollution Measurement", White, J. U. J. Advances in Environmental Science and Technology 1971, 32, 285-288. The White cell contained mirrors within the actual cell itself. The cell increased effective path length using multireflection by these mirrors. This effectively increased the sensitivity of absorption detection. However, this cell is too large for CZE purposes. It can be used only for analysis of low-level gaseous substances.
It would be advantageous to apply a multireflection technique to increase path length to CZE and, therefore, increase detection sensitivity.
It would also be advantageous to increase path length without increasing capillary size.
It would further be advantageous to create a system that is compatible with the standard CZE system in the industry.
It would further be advantageous to provide means for positioning the capillary cell with respect to an electromagnetic energy source.
It would further be advantageous to provide a reflective mechanism external to the capillary.